The goal of this research project is to understand the fundamental mechanisms by which single-subunit RNA polymerases catalyze transcription. Single-subunit RNA polymerases are widespread in nature transcribing not only the genomes of phages but also the genomes of mitochondria that are vital for energy production and survival of eukaryotes including humans. Understanding the molecular mechanisms of gene expression by single-subunit RNA polymerases has applications in developing therapeutics for numerous mitochondrial related human diseases as well as combating parasitic infections. It is recognized that transcription initiation, elongation, and termination all play essential roles in regulating gene expression. However, we lack quantitative description of these conserved transcriptional processes. By choosing enzymatically tractable systems, such as T7 RNA polymerase and mitochondrial RNA polymerase of the yeast, we are able to obtain in depth knowledge of each stage to ultimately develop a predictive and quantitative model of transcription. There is a need to develop new methods to assay the various steps of transcription initiation and elongation in real time that identify key intermediates and measure the rate constants and energetics of the elementary steps. One of our goals is to develop fluorescence-based transient state kinetic approaches that can be used in ensemble and single molecule experimental set up to measure the dynamics of transcription in real time. These methods will be developed through studies of T7 RNA polymerase and applied to understand the mechanism of mitochondrial RNA polymerases and associated factors. Unlike T7 RNA polymerase, the mitochondrial RNA polymerases require transcription factors, and one of our goals is to understand how these accessory factors aid in transcription and its regulation. The proposed studies will determine 1) the mechanism of polymerase translocation and fidelity of transcription elongation, 2) role ofthe transcription factors and accessory factors in various steps of transcription by the mitochondrial RNA polymerases, and 3) role of transcription in the initiation of replication.